Team:Groningen/Labwork/29 July 2013

Mirjam

Run the samples of the colony PCR of BBa_K823823 combined with the LacI promoter made on 27-07 on gel (order 1-8).
There are no bands around 1500 bp.

Run the purified PCR product of spec on gel.
The purified spec shows a nice band.

Run the PCR products of silk 2 on gel to do gel purification.
Only one of the tubes showed a band around the expected height.

Run the PCR products of silk 3 on gel to do gel purification
No bands appeared at the expected height.

Made a double restriction digestion with fast restriction digest for Pdes and CheY with respectively EcoRI and BamHI; and BamHI and PstI. Only a band appeared for Pdes.

Made a fast restriction digestion for RFP and eYFP and LacI+BBa_k823823. This time a control mechanism is used. So the fluorescent proteins are cut with EcoRI, PstI and EcoRI&PstI. As control also uncut eYFP is loaded on gel. It is noticed that the cut with EcoRI shows bands. When PstI is added, the bands disappear.
So a new reaction digestion is made. This time using the EcoRI and the PstI from NEB. The restriction for LacI+BBa_k823823 is made and it works. As the restriction for RFP and eYFP also works, but in very low concentrations. The bands are cut to do gel purification. (Conclusion, the PstI that we earlier used did not work correctly).

Chaline

Made a ligation for the knockout system of Des, with Des UP, Tet and Des Down.

Chaline & Mirjam

Made a PCR reaction on the ligation of knockout Des, with an annealing temperature of 55°C and an expected size of 3200 bp. Gel examination revealed that only a smear appeared.

Friso

Made a restriction digestion for Spec purified with EcoRI and BamHI.

Claudio

Two GFP reporters are picked from the iGEM database: BBa_E0840 and BBa_E0240 (backbone: pSB1C3). Those are transformation into E. Coli DH5Α (Cm resistance).

The plasmid containing mKate2 (red fluorescent protein into pGEM-T Easy vector) is inoculated in LB + Ampicillin resistance.

The colony PCR which failed on Saturday is performed again on 4 colonies (the same which were also tested the previous time) changing the annealing temperature to 46°C.
The four samples are loaded on gel and bands are spotted around the expected height (~1500 bps).
The transformation worked.